The estrogen receptor a protein (ER) is found in two-thirds of newly diagnosed breast cancers. Though ER is required for the development of breast cancer, it is paradoxically associated with a better prognosis compared to ER negative tumors, through suppression of the oncogenic transcription factor, NFkB. Our preliminary data define the molecular basis for this phenomenon. We demonstrate that ER is recruited to the NFkB response element of the Monocyte Chemoattractant-1 (MCP-1) gene in the MCF7 breast cancer cell line, and that the CBP co-activator is a bridging factor between ER and NFkB. However, the structural features that define specificity for the NFkB pathway for ER ligands are not known. We are using novel high throughput structural biology tools as a unique approach to dissecting this poorly understood signaling pathway. Chemical biology probes specific for this pathway are not available, and will provide a critical complement to our ongoing molecular and structural studies. This proposal plans to develop assays for estrogen receptor ligands that suppress NFkB without activating estrogen responsive gene expression. This will be pursued through the following: Specific Aim 1: Develop stable cell lines for luciferase assays, including an NFkB-luciferase reporter with ER expression vector, and an estrogen-responsive luciferase reporter with ER expression vector. Stable cell lines will allow mix and measure assays appropriate for HTS, and improve robustness and reproducibility. Specific Aim 2: Develop a robust and highly reproducible assay to identify ER ligands that suppress NFkB luciferase activity without activating estrogen responsive luciferase activity. Combining these screens with a commercially available ligand binding assay will allow us to find highly specific chemical probes for this signaling pathway. The results of this proposal will be assays ready for conversion to an HTS format, which can be accomplished in collaboration with the ultra-high-throughput screening group at Scripps Florida, a part of the Molecular Libraries Screening Center Network. The assay plan is to take compounds that suppress NFkB and perform the following secondary screens: identify compounds that suppress NFkB only in the presence of ER; identify compounds that do not activate estrogen responsive luciferase activity; and identify compounds that do bind ER in the commercially available ligand binding assay. Optimized chemical probes will be used for structural biology experiments to understand the mechanisms through which ER suppresses NFkB.